Our studies are directed toward establishing whether the current concepts of steroid hormone action involving free diffusion from blood into cells and binding to receptors are correct. Studies of hyperestrogenic postmenopausal women with endometrial cancer revealed that they have a 2-\to 3-fold increase in plasma-free estradiol as compared to hypoestrogenic women with osteoporosis. The absolute concentration of free estradiol (E2) is about 10[unreadable]-12[unreadable] M, which is far below the reported dissociation constant of the estrogen receptor (K[unreadable]D[unreadable] = 10[unreadable]-9[unreadable] -\10[unreadable]-10[unreadable] M). Since these values are probably too high because of errors associated with current methods of receptor analysis based upon bound hormone, we have developed a new approach involving measurement of free ligand. Recent results indicate that the dissociation constant for binding of estradiol to the estrogen receptor is 10[unreadable]-11[unreadable] M. Together with the observation that the binding reaction in uterine cytosol exhibits positive cooperativity, these results are consistent with the concept that free and albumin-bound E[unreadable]2[unreadable] are available to target cells. We are also comparing steroid binding in serum and cells from primates, including new world monkeys, that have extraordinarily high circulating levels of glucocorticoids, estrogens, and androgens. The results of these studies suggest that a steroid bound to the specific blood transport proteins (CBG and SHBG) may be the biologically important fraction. Studies of steroid metabolism in squirrel monkey cells and tissues indicate that the elevated plasma levels are the consequence of reduced clearance rather than increased secretion. Since only slight differences have been found in receptor binding characteristics, these results are incompatible with current concepts of steroid hormone action. (D)